The present invention relates to the synthesis of bioactive ceramic templates for optimum in vitro formation of bone and bone-like tissue, and the use of bioactive substrates for the enhanced cellular attachment and function of anchorage-dependent cells. The fundamental mechanisms by which material surfaces elicit the in vitro response from bone cells are optimized, thereby leading to high formation rates of extracellular material with typical characteristics of bone tissue.
Materials were first used to provide structural support during healing of bones, or to replace damaged or diseased bone tissue. Historically, the most important material selection criterion was inertness. It was believed that the implant material should only provoke the slightest reactions in the body. It is important to realize, though, that no matter how chemically inert a material may be, it always provokes a reaction upon implantation. The intensity of the reaction depends not only on the surface and bulk properties of the implant material, but also on the trauma at the time of surgery, the site of implantation, and the relative motion at the tissue-implant interface. This observation has prompted the use of xe2x80x9cbioactivexe2x80x9d materials instead of so-called inert materials. The implication is that a bioactive material must provoke a beneficial tissue response, specifically it must elicit the formation of the normal tissue at its surface and create an interface that promotes long functional life. Whereas the field of calcified tissue reconstruction has achieved this goal, this advance is not an end state, but merely a stepping stone for an even more ambitious goal: the creation of materials that are capable of serving as templates for in vitro bone tissue formation. This is part of the true future of biomaterials: creating materials that, once inserted into the body, regenerate tissues rather than replace them.
Cell culture studies with osteoprogenitor cells or cells of osteoblastic phenotype have been performed, but never achieved acceptable results. Some of these prior studies did not seek the optimization per se of extracellular material synthesis. Some prior studies used osteoprogenitor cells present in bone marrow extracts. Regardless of whether focus is placed on the determination of osteoblastic phenotype expression or elsewhere, these results can be used to determine whether one of the resultant phenomena of osteoblastic phenotypic activity was extensive or not.
It is known to obtain bone marrow cells from the femora of young adult male Wistar rats by washing them out with xcex1 MEM (minimal essential medium) supplemented with 15% fetal bovine serum, freshly prepared ascorbic acid, sodium xcex2-glycerophosphate, dexamethasone (DEX) and antibiotics. See, Davies et al. xe2x80x9cEarly extracellular matrix synthesis by bone cells,xe2x80x9d Bone-Biomaterials Workshop, J. E. Davies Ed., University of Toronto Press, (December 1990). A quantity of this cell suspension, e.g., 30 ml, containing cells from two femora, is aliquoted on to the material substrate. In a humidified 95% airxe2x80x945% CO2 atmosphere the culture is maintained for a minimum of two weeks. It was shown that a calcified matrix of globular accretions, also containing sulphur, is formed. This layer was typical for reversal lines in bone tissue, the cementum layer, and was considered by the authors as evidence that the calcified layer is the result of the expression of the osteoblastic phenotype by the cultured cells. Subsequently, there was what was called xe2x80x9cfrank bone formation.xe2x80x9d Thus, matrix production can start within a time period of intermediate duration (17 days) by differentiating bone-derived cells in vitro. It has also been reported, however, that no calcified tissue formation has been obtained on porous ceramics. See Uchida et al. xe2x80x9cGrowth of bone marrow cells on porous ceramics in vitro,xe2x80x9d J. Biomed, Mat. Res. 21:1-10 (1987). The observation in the prior art with respect to the intrinsic capability of cells to deposit a cement-like line is in any event certainly correct. The cell culture method described above is derived form Maniatopulos et al.""s xe2x80x9cBone formation in vitro by stromal cells obtained from bone marrow of young adult rats,xe2x80x9d Cell Tissue Res. 254:317-330 (1988), wherein this particular cellular activity was shown to be present in cultures without any tissue stimulating biomaterial.
The effect of porous calcium phosphate ceramic on growth and hormonal response of periosteal fibroblasts, osteoblasts, and chondrocytes has been disclosed by other workers. See, e.g., Cheng et al., xe2x80x9cGrowth of osteoblasts on porous calcium phosphate ceramic: an in vitro model for biocompatibility study,xe2x80x9d Biomaterials, 10, 63-67 (1989). As reported in this reference, the number of these cells increased 29-, 23- and 17-fold during a ten week time period. Osteoblasts retained their phenotypic expression by producing only Type I collagens. Previously, Cheng had shown that the phenotypic expression of canine chondrocytes had been retained up to 13 months when cultured on porous hydroxyapatite ceramic granules. See Cheng, xe2x80x9cIn vitro cartilage formation on porous hydroxyapatite ceramic granules,xe2x80x9d In Vitro Cellular and Developmental Biology, 21:6, 353-357 (1985). The elaboration of extracellular matrix reportedly started to appear at week one and increased throughout a thirteen month period.
Still others have studied the attachment and subsequent growth of V79 cells in contact with various calcium phosphate ceramics and found that cell growth was markedly inhibited by hydroxyapatite, and slightly inhibited by tricalcium phosphate and glass ceramics. See Katsufumi et al., xe2x80x9cThe influence of calcium phosphate ceramics and glass ceramic on cultured cells and their surrounding media,xe2x80x9d J. Biomed Mat. Res., 24:1049-1066 (1989). Under conditions of phagocytosis of small bioactive ceramic powders, RNA transcription and protein synthesis of osteoblast populations have been stimulated. See Gregoire et al. xe2x80x9cThe influence of calcium phosphate biomaterials on human bone cell activities: An in vitro approach,xe2x80x9d J. Biomed Mat. Res. 24:165-177 (1990). This phenomenon has also been observed for phagocytosing fibroblasts. It has been suggested that the increase of 3H-thymidine incorporation into DNA and the decrease of alkaline phosphatase activity probably resulted from secondary calcium messenger pathways. See Orly et al. xe2x80x9cEffect of synthetic calcium phosphate on the 3H-thymidine incorporation and alkaline phosphatase activity of human fibroblasts in culture,xe2x80x9d J. Biomed Mat. Res. 23:1433-1440 (1989). Another study by Puleo et al., xe2x80x9cOsteoblast responses to orthopaedic implant materials in vitro,xe2x80x9d J. Biomed Mat. Res. 25:711-723 (1991), provided inconclusive results regarding osteoblast attachment, osteoblast proliferation and collagen-synthesis.
Another set of studies performed in vivo, documenting materials-dependent tissue response patterns are noteworthy. A series of experiments with porous hydroxyapatite and bone marrow cells was started by Ohgushi, Goldberg and Caplan and subsequently continued separately by Ohgushi and associates in Nara, Japan and Caplan and associates in Cleveland, Ohio (USA). See Ohgushi et al. xe2x80x9cHeterotopic osteogenesis in porous ceramics induced by marrow cells,xe2x80x9d J. Ortho. Res., 7:568-578 (1989). These experiments demonstrate that the osteoprogenitor nature of the cells of a marrow cell suspension, implanted in heterotopic sites, are activated more readily when the suspension is infused into porous hydroxyapatite than when implanted by itself.
Finally, U.S. Pat. No. 4,609,501xe2x80x94Caplan et al. discloses the stimulation of bone growth that includes the in vitro exposure of isogenic fibroblasts to a soluble bone protein capable of stimulating a chondrogenic response. The exposed cells are combined with a biodegradable carrier such as fibrin, although it is also suggested that the exposed cells may also be incubated with a prosthesis. A related Caplan et al. patent, U.S. Pat. No. 4,609,551, discloses techniques for delivering the bone protein to anatomical sites, while U.S. Pat. No. 4,608,199 also to Caplan et al. discloses processes for obtaining suitable bone protein.
The present invention is focused on substrate materials and shows that appropriate modification of the material used as the substrate can lead to major differences in amount and rate of tissue formation in vitro. It is thus an object of this invention to synthesize and treat bioactive materials which can serve as the ideal templates upon which life processes, for example, bone tissue formation, can thrive.
These and other objects are met by the synthesis of porous glass without producing significant crystallization and the conditioning of the glass surface such that cell attachment is enhanced and extensive extracellular matrix (ECM) formation can take place. Control of the bioactivity reactions to produce pH variation in solution within boundaries that do not kill the cells once seeded on the porous glass is also disclosed.
Generally, the present invention discloses a bioactive material surface treated to enhance bone cell attachment and activity when the material is placed in a tissue culture medium, such that when inoculated with cells, bone tissue forms in vitro. Preferably, the bioactive comprises a glass treated to control pH, most preferably to less than 7.6. In a preferred embodiment, the bioactive material is a non-crystalline glass consisting essentially of: SiO2; CaO; Na2O; and P2O5 having the following preferred composition: 45% by weight SiO2; 24.5% by weight CaO; 24.5% by weight Na2O; and 6% by weight P2O5. Alternatively, in other preferred embodiments, the bioactive material of the present invention comprises a ceramic. Compositions such as disclosed in U.S. Pat. No. 4,478,904; PCT/US94/13152; and Paul Ducheyne, xe2x80x9cBioglass Coatings and Bioglass Compositions,xe2x80x9d J. Biomedical Materials Research, 19:273-291, 1985 (all references hereby incorporated by reference) are also contemplated. In either glass or ceramic forms, however, the material is preferably porous, and the porosity is between about 20-30% and the pore size range is about 75-200 xcexcm.
The present invention therefore discloses methods of forming a porous glass substrate comprising the steps of melting an admixture consisting essentially of SiO2, Na2O, CaO and P2O5 and then quenching the melted admixture to create a glass frit. A glass powder is formed from the glass frit that preferably has a particle size between about 40 to 70 xcexcm. A porous glass substrate is then formed from this powder by a foaming process. An important aspect of the present invention is that the resulting porous glass exhibits substantially no crystallization. Crystallization can occasionally be acceptable, but its main drawbacks are the nonuniform corrosion rates of the surface, and therefore the spatial variation of the reaction layers. Furthermore, the corrosion reactions are considerably slower, thereby necessitating long conditioning and incubation times. Alternatively, the porous glass substrate can be formed by creating a slurry of glass powder and a binder such as polyvinyl alcohol and pouring the slurry into a mold, drying the slurry and sintering it to produce a porous glass substrate. Finally, the porous glass substrate can be formed by first quenching the admixture so that a glass is formed.
This glass is transformed into a glass ceramic having more than one crystalline phase and then preferentially dissolved to result in a porous material. The preferential dissolution may be accomplished by, for example, adding a solvent such as an acid or a base.
The present invention thus discloses the formation of a porous, preferably non-crystalline, bioactive glass. It is anticipated, though, that any known bioactive glass or ceramic can suitably be transformed to become a template for in vitro synthesis of bone tissue. Inoculation times will probably differ substantially. The glass material disclosed herein can be formed into a prosthetic article such as a disk, a sleeve, a rod or any other form of substrate. Alternatively, the glass formed herein can be provided in the form of particles.
In order to enhance bioactivity and bone formation using the glass of the present invention, the glass must be treated to prepare its surface for cell attachment and to control pH prior to its inoculation with cells. Therefore, the present invention also discloses methods of forming bone tissue comprising the steps of providing a porous bioactive template consisting of the glass material described above, immersing the template in a buffer solution and immersing the template in a tissue culture medium to produce a surface which significantly enhances bone cell activity when cells are inoculated on this surface. Finally, the template is inoculated with cells and bone tissue is permitted to form thereon. Most preferably, the buffer solution is buffered at a pH of about 6.8 and the tissue culture medium has a pH not exceeding about 7.6. The cells inoculated on the template may be chosen from any preparation providing osteoprogenitor cells, bone marrow cell preparations, cells of osteoblastic phenotypic potential or of osteoblastic phenotype. Inoculation may also proceed with fibroblasts and chondrocytes.
The bioactive materials can also be treated to achieve an enhanced conformational state of subsequently adsorbed adhesion molecules and greater attachment strength of anchorage-dependent cells in general. As is discussed in more detail below, greater attachment strength signifies better cellular response, in vitro and in vivo. The treatment involves immersion in a first aqueous solution containing a concentration of ions typical of interstitial fluid followed by at least a second immersion in an aqueous solution containing at least one adhesion molecule.